DESCRIPTION: (Applicant's Description) The cause of breast cancer, the most common malignancy diagnosed in American women, remains largely unknown. Nevertheless, several genomic alterations have been detected in breast lesions. The most relevant among them are DNA amplification, loss of genetic material, and genetic instability of the abundant highly polymorphic short nucleotide repeat sequences or microsatellite instability (MSI), which in some cases are a manifestation of errors in DNA mismatch repair (MMR). It is not known, however, whether these genomic changes are responsible of the initiation of the disease, or just a consequence of chromosomal destabilization caused by tumor cell proliferation. We postulate that if the genomic alterations reported in breast cancer playa role in the initiation of the disease they should have emerged in the early phases of cancer development. In order to determine whether specific genomic alterations emerge during the initiation of the disease, and whether they differ from those expressed in later phases of cancer progression, we have developed an in vitro-in vivo experimental model of induction of cell transformation by in vitro treatment of human breast epithelial cells (HBEC) with benz (a) pyrene (BP). In this model, we have demonstrated that MSI was manifested at the stage of cell immortalization, and at early stages of cell transformation, whereas loss of heterozygosity was a late manifestation appearing in relation to the expression of tumorigenesis. The relevance of these findings lies in the fact that the loci affected by MSI in chemically transformed cells were similar to those found in spontaneously developing preneoplastic and neoplastic lesions of the human breast. Using our experimental system we will search for changes in known MMR genes at the messenger RNA and/or protein levels. Altered genes will be characterized by mutational analysis utilizing techniques such as single strand conformation polymorphism (SSCP), direct sequencing of specific mismatch repair genes, and measurement of DNA heteroduplex repair in cell extracts. Results obtained with these studies will provide functional evidence of whether the MMR mechanism is defective in cancer initiation. The knowledge gained through this exploratory work will establish the basis for more in dept studies in the role of mismatch repair in breast cancer.